Multicellular organisms have a large number of specialized organs and tissues that are assembled to form a functional unit. Coordination of various parts of an organism is achieved by chemical messenger substances termed hormone. Plant hormones are naturally occurring substances, effective in very small amounts that act as signals to stimulate or inhibit growth or regulate development. Nowadays, the following molecules are generally recognized to be plant hormones: auxins, gibberellins, cytokinins, abscisic acid, brassinolide, and ethylene.
In animals, hormones are usually synthesized in special glands and distributed via the bloodstream within the organism. Thus, they reach the target site and responsive tissues that are ready to react. There they trigger specific regulatory processes. This classical concept of hormones that was originally developed for animals was extended to higher plants. In many cases, plant hormones are active in specific target tissues, which are often different from the tissues in which the hormone is produced. However, all the plant hormones can also be detected in many tissues of the multicellular plant. This indicates that there is frequently no obligatory division between the site of synthesis and the site of action of plant hormones. If required, they are able to act on the same cells (tissues) in which they were synthesized. Thus, understanding the control of plant hormone synthesis is important in determining the relationship between synthesis and action.
Gibberellins (GAs) were originally discovered as phytotoxins in 1920s by Japanese phytopathologists. The pathogenic fungus, Gibberella fujikuroi, infects rice plants and secretes a compound that causes pathological longitudinal growth (Bakanae “mad seedling disease”). Between 1935 and 1938 the active substance was isolated and crystallized. It was called “gibberellin.” Later researches showed that GAs are also synthesized by higher plants and are very important in the regulation of growth and in differentiation processes.
The basic structure of approximately 80 GAs identified up to 1992 is the tetracyclic ring system of the ent-gibberellan (FIG. 1a). GAs contain diterpenoid carboxylic acids produced mainly from mevalonic acid via cyclization of geranylgeranyl pyrophosphate (FIG. 1b). Most of the GAs are inactive in promoting plant development. In many plants, biologically active GAs, which act as plant growth regulators, are GA1 and GA4. They can control various developmental processes, including seed germination, stem elongation, flowering, and fruit development. Thus, various modified plants that are industrially useful can be generated by modifying GA biosynthesis.
The role of GAs as mediators of environmental stimuli has been well established. Physical factors, such as light and temperature, can modify GA metabolism by changing the flux through specific step in the pathway. For example, light quality (red or far-red) and intensity (high or low) affects GA biosynthesis. In lettuce seeds and cowpea epicotyls, 3-beta hydroxylation of GA20 is enhanced by treatment with far-red light (Toyumasu et al., (1992) Plant Cell Physiol. 33, 695–701). In addition, when pea seedlings are grown in low irradiance (40 μmol/m2s), GA20 content increases sevenfold compared with plants grown in high irradiance (386 μmol/m2s), whereas in plants grown in the dark, the GA20 content is reduced as compared to that in high irradiance (Gawronska et al., (1995) Plant Cell Physiol. 36, 1361–1367).
In spite of many attempts to implicate GA metabolism in phytochrome-mediated or light intensity-mediated changes in growth rate, supporting evidence is sparse. The mechanism(s) underlying these regulatory processes will eventually be understood as a result of the current advances in the molecular biology of GA biosynthesis.
Despite considerable efforts, the site of synthesis of bioactive GAs and their mode of action in specific cells and tissues has not been clarified. Based on experiments in which plants were supplemented with 14C-labelled GAs, it is thought that they are translocated in a non-polar manner throughout the plant. More recently, grafting experiments with dwarf and wild-type pea plants indicated that GA1, one of the bioactive GA, is not transported, unlike its precursor, GA20, (Proebsting et al. (1992) Plant Physiol. 100, 1354–1360; Reid et al., (1983) J. Exp. Bot. 34, 349–364). Quantitative analyses using GC-MS and bioassays with dwarf pea plants have revealed that GAs are mainly present in actively growing and elongating tissues such as shoot apices, young leaves, and flowers (Jones and Phillips, (1966) Plant Physiol. 41, 1381–1386; Potts et al. (1982) Physiol. Plant, 55, 323–328: Kobayashi et al., (1988) Agric. Biol. Chem. 52, 1189–1194). However the exact amount of each GA in a specific tissue is difficult to determine because most GAs are present in very small amounts and most are not bioactive. Therefore, a new approach is needed to clarify the location of synthesis of bioactive GAs.
According to progress in molecular biology and genetic engineering, almost of the genes encoding GA biosynthetic enzymes so far have been cloned from various plant species. The studies of these clones have shown that GA responsive dwarf mutants lack the respective GA biosynthetic enzymes (FIG. 1b). Their expression profile indicates that the pathway is strictly regulated during development. Among these genes, GA1 from Arabidopsis, which encodes copalyl diphosphate synthase (CPS), an enzyme active early in GA biosynthesis, is highly expressed in rapidly growing tissues, e.g., the shoot apex, root tips, and flowers (Silverstone et al., (1997) Plant J. 12, 9–19). GA C-20 oxidase, which catalyzes a late step in the GA biosynthetic pathway and constitutes a small gene family, is specifically expressed in stems and developing seeds of Arabidopsis, pea, and bean where GA is required for development. They are negatively regulated by treatment with GA3 (Phillips et al., (1995) Plant Physiol. 108, 1049–1057; Garcia-Martinez et al., (1997) Plant Mol. Biol. 33, 1073–1084).
These observations lead the present inventors to speculate that GA action in various organs may depend upon the amount of endogenous GA present, which in turn depends on the regulation of expression of GA biosynthetic enzymes, rather than on the translocation of bioactive GA to the site of GA action. However, analysis of the expression of CPS or GA C-20 oxidase provides no direct evidence of the site of synthesis of bioactive GAs and regulation of bioactive GA levels because bioactive GAs are synthesized by 3β-hydroxylation which is catalyzed by GA 3β-hydroxylase.
As described above, 3β-hydroxylase catalyzes the conversion of the GA20 and GA9 to GA1 and GA4, respectively, at the final step in the synthesis of bioactive GAs (FIG. 1b). The enzymology of 3β-hydroxylase has not yet been completely clarified. However, the 2-oxoglutarate-binding region is essential for its activity, indicating that GA 3β-hydroxylase has the typical properties of a 2-oxoglutarate-dependent dioxygenase. Certain GA 3β-hydroxylase may be multifunctional; the enzyme from pumpkin endosperm catalyzes both 2β and 3β hydroxylation (Lange et al., (1997) Plant Cell, 9, 1459–1467). Maize dwarf-1,3β-hydroxylase has also been considered to be multifunctional and it catalyzes three hydroxylation steps in the maize GA biosynthetic pathway (Spray et al., (1996) Proc. Natl. Acad. Sci. 93, 10515–10518). But the nature of these GA 3β-hydroxylases is still not well known.